Extra c-myc oncogene copies in high risk cutaneous malignant melanoma and melanoma metastases.

Amplification and overexpression of the c-myc gene have been associated with neoplastic transformation in a plethora of malignant tumours. We applied interphase fluorescence in situ hybridization (FISH) with a locus-specific probe for the c-myc gene (8q24) in combination with a corresponding chromosome 8 alpha-satellite probe to evaluate genetic alterations in 8 primary melanomas and 33 advanced melanomas and compared it to 12 melanocytic nevi, 7 safety margins and 2 cases of normal skin. Additionally, in metaphase spreads of 7 melanoma cell lines a whole chromosome 8 paint probe was used. We investigated the functionality of the c-myc gene by detecting c-myc RNA expression with RT-PCR and c-myc protein by immunohistochemistry. 4/8 primary melanomas and 11/33 melanoma metastases showed additional c-myc signals relative to the centromere of chromosome 8 copy number. None of the nevi, safety margins or normal skin samples demonstrated this gain. In 2/7 melanoma cell lines (C32 and WM 266-4) isochromosome 8q formation with a relative gain of c-myc copies and a loss of 8p was observed. The highest c-myc gene expression compared to GAPDH was found in melanoma metastases (17.5%). Nevi (6.6%) and primary melanomas (5.0%) expressed the c-myc gene on a lower level. 72.7% of the patients with c-myc extra copies had visceral melanoma metastases (UICC IV), patients without c-myc gain in 35.0% only. The collective with additional c-myc copies also expressed the gene on a significantly higher level. These results indicate that a c-myc gain in relation to the centromere 8 copy number might be associated with advanced cutaneous melanoma.

Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH.

Structural chromosome aberrations (SCAs) are sensitive indicators of a preceding exposure of the hematopoietic system to ionizing radiation. Cytogenetic investigations have therefore become routine tools for an assessment of absorbed radiation doses and their biological effects after occupational exposure or radiation accidents. Due to its speed and ease of use, fluorescence in situ hybridization (FISH) with whole chromosome painting (WCP) probes has become a method of choice to visualize SCAs. Until recently, this technique was limited to a rather small number of chromosomes, which could be tested simultaneously. As a result, only a fraction of the structural aberrations present in a sample could be detected and the overall dose effect had to be calculated by extrapolation. The recent introduction of two genome-wide screening techniques in tumor research, i.e., Spectral Karyotyping (SKY) and multicolor FISH (mFISH) now allows the detection of translocations involving any two non-homologous chromosomes. The present study was prompted by our desire to bring the power of mFISH to bear for the rapid identification of radiation-induced SCAs. We chose two model systems to investigate the utility of mFISH: lymphocytes that were exposed in vitro to 3 Gy photons and single hematopoietic progenitor cell colonies isolated from a Chernobyl victim 9 years after in vivo exposure to 5.4 Sv.In lymphocytes, we found up to 15 different chromosomes involved in rearrangements indicating complex radiation effects. Stable aberrations detected in hematopoietic cell colonies, on the other hand, showed involvement of up to three different chromosomes. These results demonstrated that mFISH is a rapid and powerful approach to detect and characterize radiation-induced SCAs in the hemopoietic system. The application of mFISH is expected to result in a more detailed and, thus, more informative picture of radiation effects. Eventually, this technique will allow researchers to rapidly delineate chromosomal breakpoints and facilitate the identification of the genes involved in radiation tumorigenesis.

c-MYC and nodular malignant melanoma. A case report.

BACKGROUND: The incidence of skin cancer has been rising since the 1950s. About 75% of skin cancer-associated deaths are caused by malignant melanoma. Nodular malignant melanoma accounts for 20% of melanocytic malignant tumors and is associated with a relatively poor prognosis. Extensive research has been undertaken, but a molecular marker that can predict a more aggressive course of melanoma still has not been found. METHODS: The authors applied cytogenetic and molecular genetic techniques to a case of nodular malignant melanoma. They used comparative genomic hybridization (CGH) to identify chromosomal regions affected by genomic changes and interphase fluorescence in situ hybridization (FISH) on touch preparations of the tissue to elucidate the CGH findings further. To investigate the functionality of the affected c-MYC gene, the authors detected its transcript via reverse transcription and polymerase chain reaction. RESULTS: CGH revealed a copy number gain in the 6p and 8q24-8qter region. FISH with c-MYC and centromere eight specific probes revealed that the tumor, in contrast to unaffected skin, was characterized by a gain in copy numbers of the c-MYC gene. The c-MYC gene transcript was detected at higher levels in the tumor than in the tissue taken from the safety margin. CONCLUSIONS: The WAF1 gene located on chromosome 6p, which in this case had a copy number gain, might be involved in melanoma pathogenesis. The authors suggest that the c-MYC gene plays an important role in melanoma development and progression. The c-MYC gene seems to be affected by gaining functional copies, leading to a change in the normally regulated gene-dose effect.

Stable chromosomal aberrations in haemopoietic stem cells in the blood of radiation accident victims.

PURPOSE: The detection of long-term persistent chromosome aberrations in circulating haemopoietic stem cells after accidental radiation exposure. MATERIAL AND METHODS: Peripheral blood samples from highly exposed persons were collected 7-25 years after the radiation accidents in Moscow (1971), Kazan (1975) and Chernobyl (1996). Haemopoietic blood stem cells were analysed when investigating individual colonies derived from haemopoietic progenitor cells: burst-forming units-erythroid (BFU-E), granulocyte-macrophage-colony-forming cells (GM-CFC) and multipotent granulocyte-erythrocyte-macrophage- megakaryocyte-colony-forming cells (GEMM-CFC). Colony formation was obtained in methylcellulose cultures. Chromosome preparations in single colonies were performed using a microtechnique. RESULTS: Nine patients were investigated at 1 to 4 follow-up time points after radiation exposure. Three hundred and thirty-four single colonies were analyzed resulting in 1375 mitoses. It was found that colonies showed chromosome aberrations (ChA) up to 25 years after radiation exposure by classical cytogenetics and by fluorescence in situ hybridization (FISH). Stable aberrations were detected in 21% of colonies. They were clonal in 19% of colonies, i.e. the same abnormality was found in all cells derived from a single colony. In 2% of colonies ChA were stable but non-clonal; unstable ChA were not observed. CONCLUSIONS: The results indicate that blood-derived haemopoietic stem cells may serve as a biological indicator to detect radiation-induced ChA. Since they are considered to be in dynamic and functional exchange with stem cells in the medullary sites of blood cell formation such as bone marrow, the use of blood stem cells as a marker of radiation effects should be explored to assess the repair status of the stem cell pool as such.

Receptor tyrosine kinase and p16/CDKN2 expression in a case of tripe palms associated with non-small-cell lung cancer.

BACKGROUND: Tripe palms is a descriptive term for a cutaneous paraneoplastic keratoderma. Tripe palms are frequently associated with gastric and pulmonary carcinoma. The pathogenetic mechanism remains unknown. OBJECTIVE: To determine the influence of receptor tyrosine kinases, which are both expressed in pulmonary carcinomas and in human skin, we performed expression studies on epidermal growth factor receptor (EGFR), HER2, HER3 in a skin sample of tripe palms obtained from a patient with non-small-cell lung cancer with lymph node involvement. Two months after diagnosis, the patient had developed palmoplantar 'tripe palms'. Additionally, the expression of SRC, c-myc and p16/ CDKN2 were studied. METHOD: Conventional reverse-transcription polymerase chain reaction was performed on a tissue sample of tripe palms. RESULTS: Weak expression of HER2 and of p16/CDKN2 was found. EGFR, HER3, c-myc and SRC were not expressed. CONCLUSION: Receptor tyrosine kinases of subclass I, the tyrosine kinase SRC and the oncogene c-myc play no major role in the pathogenesis of this case of tripe palms.

Interactions between adult human prostatic epithelium and rat urogenital sinus mesenchyme in a tissue recombination model.

Tissue recombinants composed of adult human prostatic epithelium (hPrE) and rat urogenital sinus mesenchyme (rUGM) were grafted beneath the renal capsule of athymic rodent hosts. The pseudostratified human epithelium initially became multilayered, solid epithelial cords emerged, grew into the surrounding mesenchyme and canalized to regenerate a pseudostratified epithelium. Basal cells expressed cytokeratins 5 and 14, while luminal cells expressed cytokeratins 8 and 18, prostate specific antigen and prostatic acid phosphatase. The rat mesenchymal component differentiated into thick sheets of smooth muscle, characteristic of the human but not the rat prostate. These findings indicate that epithelial-mesenchymal interactions were reciprocal. Rat UGM induced adult hPrE to form new ductal-acinar tissue, involving epithelial proliferation, ductal branching morphogenesis and functional cytodifferentiation. Concurrently the epithelium dictated smooth muscle differentiation and patterning. Species-specific reverse transcriptase polymerase chain reaction SC (RT-PCR) analysis of the tissue recombinants was performed to separately examine the expression of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), TGF-beta 1, and TGF-beta 3 in the epithelium, stroma and host components of the graft. All of these genes, except TGF-beta 1, were expressed in all three tissues. Human TGF-beta 1 was not detected, indicating that this gene was not expressed in human prostatic epithelium but was present in stroma.

Optimization of non-isotopic in situ hybridization: detection of the Y chromosome in paraformaldehyde-fixed, wax-embedded cat retina.

A technique was developed to detect the Y chromosome in paraformaldehyde-fixed diethylglycoldiesterate-embedded cat retina. The Y chromosome specific DNA probe was labeled with digoxigenin through polymerase chain reaction incorporation. After treatment of paraformaldehyde-fixed, diethylglycoldiesterate-embedded tissue sections with deoxyribonucleic acid decondensation and proteolytic digestion, non-fluorescent, non-isotopic in situ hybridization was performed on the retina sections. Most extensive treatment was required for the outer nuclear layer while the inner nuclear layer required more extensive treatment than the retinal pigment epithelial cells. Under optimal pretreatment conditions, the male cat retina displayed black spots which specifically localized at the periphery of the nuclei, while the female cat retina showed negative staining for the Y chromosome specific probe. The technique allows observation of the Y chromosome signal with preservation of retinal morphology and thus may be a valuable tool to discriminate donor cells in retinal pigment epithelial cell and photoreceptor cell transplants.

Cytogenetic and molecular genetic characterization of a chromosome 2 rearrangement in a case of human papillary thyroid carcinoma with radiation history.

Karyotype analysis of a primary culture from a case of papillary thyroid cancer (PTC) showed an abnormal short arm of one homologue of chromosome 2 as sole abnormality in 4 of 16 metaphases. Based on G-banding analysis, two different aberration types on chromosome 2 could be assumed representing either a del(2)(p22-23) or a pericentric inversion. Further comparative genomic hybridization (CGH) analysis as well as fluorescence in situ hybridization (FISH) analysis were performed to confirm the assumed alterations. While CGH analysis showed no loss of chromosome 2 material, FISH with yeast artificial chromosome (YAC) probes homologous to the region 2p22-23 demonstrated two pericentric inversions of chromosome 2 involving different breakpoints on 2p in 6.8% and 4.2% of the metaphases, respectively. Polymerase chain reaction (PCR) analysis with degenerated oligonucleotide primers that bind within the conserved catalytic domain of tyrosine kinase (tk) genes resulted in amplification products with DNA of YAC 851D11 suggesting the presence of such genes at or near the translocation breakpoint.

Immunocytochemical detection of p53 in human thyroid carcinomas is associated with mutation and immortalization of cell lines.

Mutations in the tumor suppressor gene p53 are the most-common mutations found in human cancers. In thyroid cancers, p53 mutations generally are found only in poorly differentiated and undifferentiated tumors and in cell lines. To determine the prevalence of p53 mutations in thyroid neoplasms and thyroid cell lines, we screened 58 thyroid tissues and 3 thyroid cell lines, p53 primers bracketing exons 4, 5/6, 7, and 8 were used to amplify genomic DNA using the PCR. Mutations were screened by denaturing gradient gel electrophoresis and confirmed by sequencing. The two papillary thyroid cancer cell lines and the follicular thyroid carcinoma cell line (positive control) had transitions (CGT->CAT) in exon 8, codon 273, resulting in the replacement of arginine with histidine. No normal thyroid tissues or primary tumors from which the cell lines were derived demonstrated exon 8 mutations, using this technique. p53 immunocytochemistry demonstrated a progression of p53 immunopositivity between synchronous and metachronous neoplasms, paralleling the neoplastic progression from a benign adenoma to primary carcinoma, regional, and distant metastasis and ultimately, the cell lines, where intense immunopositivity is noted. In addition, fluorescence in situ hybridization, using probes specific for the p53 locus, revealed the presence of 3 homologues of p53 in the follicular cell line and 2 homologues in the papillary and Hürthle cell lines. These results suggest that a point mutation present in a small number of original tumor cells and amplification of the mutant allele may be responsible for immortalizing well-differentiated thyroid cancer cells into cell lines.

Species-specific in situ hybridization with fluorochrome-labeled DNA probes to study vascularization of human skin grafts on athymic mice.

The skin replacements presently used for wound coverage vascularize less well than do autologous split-thickness skin grafts. Understanding how autologous grafts vascularize can lead to methods to improve skin-replacement vascularization. Fresh human skin was grafted on athymic mice. Endothelial cells were stained by immunohistochemistry. Human and mouse cells were distinguished by in situ hybridization with fluorescent species-specific DNA probes. The number of mouse and human endothelial cells in the graft were counted. Initial vascularization of the graft is limited solely to anastamosis of severed ends of human vessels in the graft-to-mouse vessels in the recipient bed. Mouse endothelial cells gradually replace human endothelial cells in the graft. This demonstrates that initial vascularization of skin grafts is solely the result of inosculation rather than neovascularization. Skin replacements that contain vessels should vascularize like skin grafts, and methods to repopulate replacements with capillaries should be sought.

Molecular and cytogenetic characterization of a t(1;10;21) translocation in the human papillary thyroid cancer cell line TPC-1 expressing the ret/H4 chimeric transcript.

BACKGROUND: Activation of the ret proto-oncogene by three different chromosomal rearrangements occurs in up to 25% of papillary thyroid carcinomas. We developed a rapid screening technique to detect ret rearrangements in human interphase and metaphase cells on the basis of multicolor fluorescence in situ hybridization (FISH) of locus-specific DNA probes. METHODS: DNA from individual clones representing the respective ends of a yeast artificial chromosome (YAC) contig spanning the entire ret gene locus were labeled with either digoxigenin (visualized in red) or biotin (green) and hybridized to normal human lymphocytes and the papillary thyroid cancer cell line TPC-1 expressing the ret/H4 chimeric transcript. Further detailed analysis was performed with whole chromosome painting probes and locus-specific probes (YACs, P1s, DNA repeat probes) on tumor metaphase spreads. RESULTS: Hybridization of the YACs to unrearranged ret loci in normal human lymphocyte interphase nuclei showed two yellow domains because of probe overlap. Hybridization to TPC-1 interphase nuclei showed one yellow domain, and 1 red and 1 green domain separated by a large physical distance. Further analysis of metaphase spreads revealed a complex translocation t(1;10;21)(1pter > 1q31::21q22.1 > 21qter; 10q11.2 > 10pter::1q31 > 1qter; 21pter > 21q22.1;;10q21.2 > 10q11.2::10q21.2 > 10qter) and loss of the H4 gene locus on the nontranslocated chromosome 10. CONCLUSIONS: Break point spanning probes can reliably detect ret rearrangements in interphase nuclei. Locus-specific and whole chromosome painting probes can be used to further characterize complex rearrangements by fluorescence in situ hybridization to metaphase spreads. The papillary thyroid cancer cell line TPC-1 carries the paracentric inversion 10q, inv(10)(q11.2q21) and a complex t(1; 10; 21) translocation. Deletion of the H4 gene on the chromosome 10 not involved in the t(1; 10; 21) translocation suggests lack of normal H4 expression in the TPC-1 cell line. Further studies will have to address the role of the H4 gene product in tumor genesis and progression.

The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1-21.2.

The interferon-inducible double-stranded RNA-specific adenosine deaminase is an RNA-modifying enzyme implicated in the generation of biased hypermutations viral RNAs and the site-selective editing of mammalian mRNAs of neural origin. The gene for the dsRNA-specific adenosine deaminase has been mapped by fluorescence in situ hybridization (FISH) of genomic clones to a single locus on human chromosome 1 bands q21.1-21.2. Simultaneous multicolor FISH including lambda clones and yeast artificial chromosomes showed a localization of the gene in band 1q21 centromeric of D1S1705.

Quantitative DNA fiber mapping.

The assembly of sequence ready, high-resolution physical maps and construction of minimally overlapping contigs for the human as well as model genomes requires accurate determination of the extent of overlap between adjacent clones as well as their relative orientation. This is presently done by procedures such as clone fingerprinting, Southern blot analysis or clone end sequencing. We present a complementary analytical technique to map directly cloned DNA sequences on to individual stretched DNA molecules. This approach uses the hydrodynamic force of a receding meniscus to prepare straight high molecular weight DNA molecules that provide a linear template of approximately 2.3 kb/microns on to which the cloned probes can be mapped by in situ hybridization. This technique has numerous advantages such as a very high density of mapping templates, reproducible stretching of the mapping template providing a linear genomic scale, determination of clone orientation and direct visualization of DNA repeats. The utility and accuracy of quantitative DNA fiber mapping are illustrated through three examples: (i) mapping of lambda DNA restriction fragments along linearized approximately 49 kb long lambda phage DNA molecules with approximately 1 kb precision; (ii) localization of the overlap between a cosmid and a colinear P1 clone; and (iii) mapping of P1 clones along an approximately 490 kb yeast artificial chromosome (YAC) with approximately 5 kb precision and estimation of the approximately 25 kb gap between them.

Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes.

Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.